L-arabitol dehydrogenase (LAD) and xylitol dehydrogenase (XDH) are involved in the degradation of L-arabinose and D-xylose, which are among the most abundant monosaccharides on earth. Previous data demonstrated that LAD and XDH not only differ in the activity on their biological substrate, but also that only XDH has significant activity on D-sorbitol and may therefore be more closely related to D-sorbitol dehydrogenases (SDH). In this study we aimed to identify residues involved in the difference in substrate specificity.Aspergillus niger on human SDH identified two residues in LadA (M70 and Y318), that may explain the absence of activity on D-sorbitol. While introduction of the mutation M70F in LadA of A. niger resulted in a nearly complete enzyme inactivation, the Y318F resulted in increased activity for L-arabitol and xylitol. Moreover, the affinity for D-sorbitol was increased in this mutant.Phylogenetic analysis demonstrated that LAD, XDH and SDH form 3 distinct groups of the family of dehydrogenases containing an Alcohol dehydrogenase GroES-like domain (pfam08240) and likely have evolved from a common ancestor. Modelling of LadA and XdhA of the saprobic fungus These data demonstrates that Y318 of LadA contributes significantly to the substrate specificity difference between LAD and XDH/SDH. L-arabinose and D-xylose are two of the most abundant monosaccharides in nature. They are components of the plant cell wall polysaccharides xylan, xyloglucan and pectin  and therAspergillus niger, Aspergillus nidulans and Trichoderma reesei (Hypocrea jecorina) and, except for L-arabinose reductase and L-xylulose reductase, all genes from the pathway have been identified and characterised [A. niger L-arabitol dehydrogenase and xylitol dehydrogenase demonstrated that L-arabitol dehydrogenase is active on L-arabitol and xylitol, but not on D-sorbitol, while xylitol dehydrogenase is active on xylitol and D-sorbitol, but not on L-arabitol [The pentose catabolic pathway has been studied mainly in cterised -11. In varabitol . In thisA. niger, A. nidulans and A. oryzae at the comparative Aspergillus server from the Broad Institute http://www.broad.mit.edu/annotation/genome/aspergillus_group/MultiHome.html. However, the highest hit for these fungi was xylitol dehydrogenase (data not shown). In addition, the KEGG website http://www.genome.ad.jp/dbget-bin/www_bget?enzyme+1.1.1.15 was searched for putative D-sorbitol dehydrogenases of A. niger. Two of these corresponded to ladA and xdhA, while a third was An09g03900. In addition, two homologues of A. nidulans ladA, ladB and ladC, have been described [ladB were only found in A. niger and A. oryzae, while orthologues for ladC were only absent in N. crassa and T. reeseii out of the 8 fungi tested in this study.To determine whether fungal genomes contain homologues of D-sorbitol dehydrogenases of higher eukaryotes, the human D-sorbitol dehydrogenase  amino acescribed  althoughA. niger gene found in the KEGG database splits of from the xylitol dehydrogenase branch, although this clade only has a low bootstrap support (50%). The ladB and ladC groups split of from the ladA branch forming clearly defined groups.To determine the phylogenetic relationships between L-arabitol dehydrogenases, xylitol dehydrogenases and D-sorbitol dehydrogenases, an alignment was performed using amino acid sequences of established and putative L-arabitol and xylitol dehydrogenases of eight fungi, D-sorbitol dehydrogenases of ten eukaryotes and the other genes found in the analysis described above. A bootstrapped NJ tree  are located close to the substrate   Table . In conter Table .Comparison of the deduced amino acid sequences of LadA and XdhA to other L-arabitol, xylitol and D-sorbitol dehydrogenases, as well as some putative dehydrogenases with unknown function demonstrated that these enzymes form distinct groups in the family of dehydrogenases containing an Alcohol dehydrogenase GroES-like domain (pfam08240). Previously it was suggested that L-arabitol dehydrogenase might be the fungal orthologue of D-sorbitol dehydrogenase of higher eukaryotes . HoweverA. niger ladA and xdhA appears highly specific for L-arabinose and D-xylose [A. niger (An09g03900) that is in the groups that splits of the XDH branch in the tree. As orthologues for this gene were found in all tested fungi, it appears to encode a conserved function. However, bootstrap support for similarity of these enzymes to SDH is weak, indicating that no reliable prediction of function is possible based on these results.Interestingly, our study suggests that there is no clear fungal orthologue of SDH, based on BLAST and KEGG analysis. As the expression of D-xylose , it is uA. nidulans [The two homologues of LadA described for nidulans  cluster nidulans , as a UVA. niger LadA and XdhA on human D-sorbitol dehydrogenase revealed a large number of amino acids that are conserved in all three types of dehydrogenases, including the residues involved in Zinc binding  [Modelling of the structure from equence) . None ofequence)  this couA. niger resulted in almost complete inactivation of the enzyme on a variety of substrates. The reason for this is not clear at this point, but a possible explanation could be that M70 in this particular enzyme influences the 3-dimensional structure; thus promoting enzyme activity. As the aim of this study was to identify residues important in substrate specificity, we did not further investigate this mutation.The M70F mutation of LadA of max and Kcat increased for L-arabitol and xylitol. Projection of the catalytic site of LAD, SDH and XDH predicts that the tyrosine residue in LAD and the phenylalanine in SDH and XDH are in exactly the same position . D-sorbitol and xylitol differ structurally from L-arabitol with respect to positioning of the OH-group on C2 and C4, while D-sorbitol has an additional OH group at C5 compared to xylitol . Transformation and purification of the recombinant proteins using Ni-agarose (Qiagen) was performed according to the supplier's instructions.Derivatives of the expression vector pQE32 containing wild type and mutated versions of + and 100 mM substrate. Reductase activities were determined using 50 mM sodium phosphate pH 7.6, 0.2 mM NADH and 100 mM substrate. Absorbance changes at 340 nm (ε = 6.22 mM-1 cm-1) were measured on a Unicam UV-1 spectrophotometer . Sheep liver SDH was obtained from Sigma (S3764).All enzyme assays were performed at 20°C. Dehydrogenase activities were determined using 100 mM glycine pH 9.6, 0.4 mM NADA. niger LadA and XdhA structures were generated using the SWISS-MODEL program http://swissmodel.expasy.org//SWISS-MODEL.html[Models of ODEL.html-21 with ODEL.html. The modODEL.html.Site directed mutagenesis was performed using the Quik Change protocol . Two complementary oligonucleotides of 30–34 nucleotides were designed for each mutation, carrying the mutation in the middle of the oligonucleotide. PCR mixtures contained 50 ng of DNA template, 125 ng of each oligonucleotide, 1 μl of a 10 mM dNTP stock, 5 μl of 10× pfu buffer, and sterile water to a total volume of 24 μl. Before the start of the PCR, 1 μl of pfu DNA polymerase (Stratagene) was added. The reaction parameters were: denaturation of the DNA for 5 min at 95°C, followed by 16 cycles of 30 s denaturation (95°C), 1 min annealing (56°C) and 15 min amplification (68°C). The product was incubated for 4 h with DpnI at 37°C. This enzyme degrades methylated (template) DNA but not the DNA amplified during the PCR.LR carried out the modelling studies. CR and BTA carried out the biochemical analysis. RPdV drafted the manuscript. All authors read and approved the final manuscript.


This study aims to elucidate the effects of chrysin on human ER-negative breast cancer cell line, MDA-MB-231. The study demonstrated that treatment of MDA-MB-231 cells with 20 μM chysin for 48 h significantly inhibited the growth of MDA-MB-231 cells and induced cytoplasmic lipid accumulation in the cells, but that the observed of cell death was not caused by apoptosis. The expression of PPARalpha mRNA in chrysin-treated MDA-MB-231 cells was significantly increased, which was likely associated to the proliferation of the cells post chrysin treatment. The cells are highly aggressive, invasive and poorly-differentiated . It is tIn this study, the viability of MDA-MB-231 cells treated with different concentrations of chrysin and for different treatment times was evaluated using trypan blue assay. Determination of the preliminary viability effect of chrysin, in this study, was sourced from previous studies described in 2.p < 0.05). Chrysin at 25 μM concentration exhibited a similar significant inhibitory effect upon the 20 μM concentration with a reduction of cell viability to 73.1% (p < 0.05). Thus, 20 μM chrysin for 48 h was used as the effective concentration and treatment time for subsequent cell differentiation and cell death experiments.Trypan blue, use in this study, is a fast and reliable colorimetric method to assess the cell viability. The assay, in which trypan blue is excluded from live cells but accumulated in dead cells, is one of the most commonly used methods for calculating the percentages of live and dead cells in a given cell population . The tryCell death, particularly apoptotic DNA fragmentation was examined in chrysin-treated MDA-MB-231 cells because the reduction of cell viability was likely to be an outcome of mitochondrial dysfunction, which would eventually lead to the cell death, according to Mosmann . Howeverp < 0.05), as reflected in the intensity of the band compared with the control samples . There was also an increase in the intensity of the PPARgamma band in chrysin-treated MDA-MB-231 cells but it was not statistically significant when compared with the control samples . This phenomenon indicates that increased expression of PPARalpha mRNA may directly or indirectly plays an important role in MDA-MB-231 cells when the cells were inhibited by chrysin treatment.Following semi-quantitative RT-PCR, the gels showed that single bands were detected for the PPARalpha, PPARdelta, PPARgamma and GAPDH bands in both the chrysin-treated and control DMSO-treated MDA-MB-231 cells . The PCRet al. .The MDA-MB-231 cell line was a kind gift from the John Hopkins Research Center. The cells were maintained with DMEM (3.2.3 cells/mL in two 24-well cell culture plates (NUNC). When the cells of each well reached approximately 70% confluency, increasing concentrations of chrysin (5–25 μM) were added and the cells were incubated for 24 h and 48 h. DMSO (0.1%) was used as diluent control in this study. All control and experimental samples were cultured in triplicate. After 24 h treatment, the cells were harvested using trypsin/EDTA solution (Gibco BRL). The harvested cell suspension (0.9 mL) from each well was added with 0.1 mL of Trypan Blue Solution (Sigma-Aldrich) and was incubated at room temperature for 5 min. Living and dead cells were then counted using a hemocytometer and the cell viability (%) was calculated as below:The effect of chrysin on the growth of MDA-MB-231 cells was determined using the trypan blue assay. For this purpose, the MDA-MB-231 cells were seeded at a density of 1.0 × 10Living cells were observed excluding the dye (unstained cells) whereas dead cells took up the blue dye. The same method was used to calculate the viability of MDA-MB-231 cells after 48 h of chrysin treatment. Analysis of one-way ANOVA was used to compare the viability of MDA-MB-231 cells in different concentrations of chrysin treatment with control DMSO for 24 h and 48 h. The lowest concentration at the effective treatment time which showed a significant reduction of cell viability was used as the effective concentration for subsequent experiments.3.3.3 cells/well in 24-well cell culture plates (NUNC). When the cells reached approximately 70% confluent, the cells were treated with increasing concentrations of chrysin for 48 h. The culture supernatants were then withdrawn for measurement of the LDH release using Roche Cytotoxicity Detection kit. The amount of LDH release in the culture supernatant was measured based on absorbance of color generated from each sample, according to the manufacturer’s instruction. The absorbance was read at 492 nm against 620 nm wavelength using ELISA reader (Thermo Labsystems). All treatments and control samples were analyzed in triplicate. The percentage of cell viability was calculated as / (High control-Low control), where low control is the absorbance of cells cultured in assay medium and high control is the absorbance of cell cultured in the presence of 1% (v/v) Triton-X. The high control was then referred as sample with 100% LDH release.The trypan blue assay was validated using LDH assay where the cells were seeded at a density of 1.0 × 103.4.4 cells/mL were seeded into 10 cm2 culture dishes for detection of apoptotic DNA fragments and extraction of total RNA for semi-quantitative RT-PCR. For detection of DNA fragmentation by fluorescence microscopy (TUNEL assay) and examination of cytoplasmic lipid accumulation, 1×103 cells/well were seeded into 4-well culture slides. The cells were grown in growth medium until they were approximately 70% confluent and then pre-incubated with assay medium for several hours before treatments. Subsequently, the effective concentration of chrysin or 0.1% DMSO were added to the cells, and the cells were incubated at 37 °C in 5% (v/v) CO2 for the selected effective treatment time. Following chrysin treatment, samples were subjected to examination of cytoplasmic lipid accumulation, detection of apoptotic DNA fragments, TUNEL assay and extraction of total RNA, as described below.After obtaining the effective concentration and treatment time for chrysin on the growth of MDA-MB-231 cells, 2 groups of cell cultures (control DMSO-treated and chrysin-treated) were prepared in triplicate for subsequent experiments. A total of 1×103.5.Cytoplasmic lipid accumulation in chrysin-treated and control DMSO-treated MDA-MB-231 cells was examined using the Oil-Red O staining. The cells grown on culture slides were fixed with Fixation Solution  before being stained with Oil-Red O solution (Sigma-Aldrich) at 37 °C for 2 h. Samples were then counter-stained with Mayer hematoxylin solution (Sigma-Aldrich) and mounted with aqueous mounting medium. The morphology of the stained cells was examined using a Nikon eclipse TS100 inverted microscope at 200× magnification and the pictures were taken with a Nikon COLPIX digital camera.3.6.Apoptotic DNA fragment detection in chrysin-treated and control DMSO-treated MDA-MB-231 cells was performed using the Calbiochem Suicide Track DNA Ladder Isolation Kit, according to the manufacturer’s instructions. For this purpose, samples grown in culture dishes were harvested and re-suspended in the lysis buffer provided in the kit. The DNA was extracted and subjected to electrophoresis on 1% agarose gels containing 0.1 μg/mL EtBr. Samples on the gel were visualized and photographed using the GeneSnap software on the Gene Genius Image Analyser.3.7.In Situ Cell Death Detection Kit, Fluorescein, according to the manufacturer’s instructions. For this purpose, samples grown on culture slides were dried and fixed with freshly prepared Fixation Solution, as described above. Cells were then incubated in a Permeabilisation Solution (0.1% Triton X-100 in 0.1% sodium citrate) before being re-suspended in the TUNEL reaction mixture provided in the kit. Samples were mounted with aqueous mounting medium and observed under a fluorescence microscope (Nikon ECLIPSE TE2000-U). DNase I recombinant was used as positive control for this assay.DNA fragmentation detection of TUNEL in chrysin-treated and control DMSO-treated MDA-MB-231 cells was performed using the Roche 3.8.Extraction of total RNA from chrysin-treated and control DMSO-treated MDA-MB-231 cells was performed using the TRI Reagent (Molecular Research Center), according to the manufacturer’s instructions. The integrity of total RNA extracted was confirmed using agarose gel electrophoresis, whilst purity and yield were measured using an Eppendorf spectrophotometer. Subsequently, 1 μg of total RNA per sample was reverse transcribed to cDNA using the Fermentas RevertAid™ H Minus First Strand cDNA Synthesis Kit, according to the manufacturer’s instructions. The success of cDNA synthesis was validated with PCR amplification using human GAPDH housekeeping gene.3.9.2, 2.0 μL of 10× Taq Buffer with (NH4)2SO4, 0.2 μL of Taq DNA Polymerase, 1.0 μL of each 20 pmol forward and reverse primers and DNase/RNase-free water. Each PCR reaction was carried out in triplicate and the reaction conditions were set as below for 35 cycles:Pre-amplification: 94 °C for 2 minDenaturation: 94 °C for 45 sAnnealing: 60 °C for 30 sElongation: 55 °C for 30 sExtension: 72 °C for 1 minTermination: 73 °C for 10 minFollowing cDNA synthesis, levels of PPAR mRNAs in chrysin-treated and control MDA-MB-231 cells were semi-quantified using PCR and gel densitometry. The levels of mRNAs were expressed as the band intensity of PPAR PCR products relative to the control GAPDH PCR product. The student’s t-test was used to compare the expression of PPAR mRNAs between chrysin-treated and control DMSO-treated cells. The primers used for the PCR reactions are detailed in The PCR products were subjected to electrophoresis on a 0.8% (w/v) agarose gel containing 0.1 μg/mL EtBr. Bands of PCR product on the gel were visualized and photographed using the Gene Genius Image Analyser, and the band intensity was analysed using the LabImage 1D 2006 Professional software (KAPELAN Bio-Imaging Solution).3.10.GraphPad software. The experiments were repeated several times to confirm the reproducibility of the results. All values are expressed as mean ± SD. A p value of less than 0.05 was regarded as statistically significant.All graph drawing and statistical calculations were performed using 4.This study identified chrysin to be a potential inhibitor on the growth of MDA-MB-231 cell line. Our study, for the first time, provides mechanistic evidence that chrysin treatment inhibits the growth of MDA-MB-231 cells and results directly or indirectly in increased expression of PPARalpha mRNA. However, further studies are required to investigate the role of PPARalpha in the death mechanism of these cancer cells induced following chrysin treatment. Such studies would help to address the hypothesis for developing effective chrysin-based for human ER-negative breast cancer treatments.


Caenorhabditis elegans is both sensitive and tolerant to hypoxic stress, particularly when the evolutionarily conserved hypoxia response pathway HIF-1/EGL-9/VHL is involved. Hypoxia-induced changes in the expression of a number of genes have been analyzed using whole genome microarrays in C. elegans, but the changes at the protein level in response to hypoxic stress still remain unclear.The nematode C. elegans. Two-dimensional difference gel electrophoresis (2D-DIGE) was used to compare the proteomic maps of wild type C. elegans strain N2 under a 4-h hypoxia treatment (0.2% oxygen) and under normoxia (control). A subsequent analysis by MALDI-TOF-TOF-MS revealed nineteen protein spots that were differentially expressed. Nine of the protein spots were significantly upregulated, and ten were downregulated upon hypoxic stress. Three of the upregulated proteins were involved in cytoskeletal function , while another three upregulated  were ATP synthases functionally related to energy metabolism. Four ribosomal proteins  were downregulated, indicating a decrease in the level of protein translation upon hypoxic stress. The overexpression of tropomyosin (LEV-11) was further validated by Western blot. In addition, the mutant strain of lev-11(x12) also showed a hypoxia-sensitive phenotype in subsequent analyses, confirming the proteomic findings.Here, we utilized a quantitative proteomic approach to evaluate changes in the expression patterns of proteins during the early response to hypoxia in C. elegans, and this information will help broaden our knowledge on the mechanism of hypoxia response.Taken together, our data suggest that altered protein expression, structural protein remodeling, and the reduction of translation might play important roles in the early response to oxygen deprivation in  Caenorhabditis elegans (C. elegans) has been proven to be an valuable model organism for studying the molecular response to hypoxia [C. elegans is sensitive to hypoxic stress, resulting in a phenotype characterized by decreased levels of feeding, movement, and oxygen consumption, it can also survive oxygen tensions between 1% and 0.2% by activating the hypoxia response pathway or anoxic conditions by entering suspended animation. Worms at all stages of development can survive at least one day of anoxia with a viability of 90% [C. elegans is relatively hypoxia-resistant compared to most mammalian cells [et al. reported that the C. elegans homolog of the HIF-1 (hypoxia induced factor-1) α subunit is hif-1 and that the HIF-1/EGL-9/VHL pathway is evolutionarily conserved [C. elegans since these small worms were generally thought to rely on diffusion from the environment for gaseous exchange due to their lack of a specialized respiratory or circulatory system [et al. identified 33 putative globin genes by a careful in silico analysis of the genome of C. elegans [C. elegans are still not completely understood. Therefore, these mechanisms should be studied to gain more knowledge on oxygen-deprivation sensing and survival in C. elegans.Hypoxic stress can induce apoptosis but also trigger adaptive mechanisms for cell survival. Mammalian cells respond to hypoxia by changes in the expression of numerous genes and proteins to increase anaerobic energy production, protect cells from hypoxic stress, and increase local angiogenesis ,2. Recen hypoxia ,4. Althoy of 90% -7, and Can cells . Powell y system ,11. Rece elegans ,13. All C. elegans to hypoxia, including its behavioral and physiological issues, has been well studied [C. elegans [et al. compared hypoxia-induced changes in mRNA expression in wild type, hif-1-deficient, and vhl-1-deficient C. elegans strains using whole genome microarrays [C. elegans, however, has not yet been performed.The response of  studied -16. More elegans -19. For roarrays , which rroarrays , other rroarrays ,23, zebrroarrays , rainbowroarrays , shrimp roarrays  and otheC. elegans, proteomic approaches are becoming part of the essential toolbox for the study of gene function [et al. also analyzed the worm proteome by 2D gel electrophoresis liquid chromatography (2D-LC) combined with electrospray ionization tandem mass spectrometry (ESI-MS/MS) and identified a total of 1,616 proteins, including 110 secreted/targeted proteins and 242 transmembrane proteins [Escherichia coli [, Saccharomyces cerevisiae [et al. compared the proteome maps from all six developmental stages, embryonic, L1-L4 larva1, and adult, to examine the expression profiles of 165 proteins during C. elegans development [In function . For exafunction  or when function . Mawuenyproteins . Quantitproteins ,32. Two-hia coli , Saccharrevisiae , mouse [revisiae  and humarevisiae . Using 2elopment .C. elegans, a 2D-DIGE and mass spectrometry (MS) based approach was used to determine the overall profile of protein expression during the early response to hypoxia. Differentially expressed protein spots were identified, and the proteins of interest were validated by Western blot. Further study of one of the hypoxia-induced proteins expressed at a high level was performed by using a mutant strain obtained from the Caenorhabditis Genetics Center (CGC). Consistent with our proteomic findings, the lev-11(x12) mutant strain had a higher death rate than the wild type strain N2 under hypoxic stress. To the best of our knowledge, this study is the first to apply a proteomic approach to screen differentially expressed proteins in response to hypoxia in C. elegans, and the results may provide new clues to understand the mechanism of hypoxia response better.To better understand the molecular mechanisms underlying the response to hypoxic stress at the protein level in C. elegans under hypoxia or normoxia (control) for 4 h. The labeled samples were separated, and the fluorescent images were obtained  were selected , identified as the most highly induced protein by hypoxia, was further examined by Western blot. Tropomyosin (LEV-11) was significantly upregulated in the 4-h hypoxia treatment samples when compared with the controls (lev-11(x12), daf-2 (hypoxia-resistant), and daf-16 (hypoxia-sensitive) were examined under hypoxic conditions . The death rate of the wild type N2 strain and lev-11(x12) mutant gradually increased as the time under hypoxic conditions increased as well as after a 24-h recovery  were compared with the lev-11 (x12) strain  after 12 h and 14 h of hypoxia treatment , respectively. When compared with the wild type worms, the lev-11 (x12) mutants have a higher death rate and are more sensitive to oxygen deprivation. After 12 h of hypoxia, the death rate of the lev-11 (x12) strain was higher than that of the daf-16 mutant. These data further support the proteomic data presented here and suggest that the protein tropomyosin (LEV-11) has an important role in the early hypoxia response and survival for C. elegans.To determine the function of tropomyosin (LEV-11), the survival ability of the L4 stage of the wild type and the mutants P = 6.54E-06). Other proteins are involved in the positive regulation of growth, ATP metabolic process, purine ribonucleotide binding, hydrogen transport, and proton transport, with significant P-values. These biological processes are all consistent with changes made in response to stress conditions. These classifications provide clues to understand better the proteomic expression changes in the nematode in vivo during the early response to hypoxia.A gene ontology (GO) classification was performed to analyze the distribution of the differentially expressed proteins identified in this study within the different biological process categories Table . The larC. elegans is a model organism for research in the fields of developmental biology, molecular biology, neurobiology and toxicology [C. elegans can also be used as a model organism for hypoxia studies. Using 2D-DIGE followed by protein identification through mass spectroscopy, nineteen proteins that responded during the early stage of hypoxia exposure were successfully identified. Although only a small number of proteins was identified, a precise evaluation of the results presented here still needs to be performed.xicology -40. HereC. elegans, and we totally used three batches of C. elegans as independent experiments to meet the standard for biological replicates. In addition, the quantitative proteomic method, DIGE, was used in this study. Briefly, samples were reverse-labeled (cy3 or cy5) to enable all comparisons and eliminate any dye-labeling bias. Samples were also mixed together and run on the same gels with an equal amount of the cy2-labeled standard. Cy2 was used as a standard on all gels to aid image matching and cross-gel statistical analyses. The use of this internal standard in all 2-DE gels greatly improved the accuracy of spot matching and the reliability of protein quantification. Therefore, this method was sufficient to enable us running two DIGE-gels at the same time, and the Decyder software was used to analyze the data. Protein spots that were differentially expressed in hypoxia-treated and control groups  were marked. Protein samples were extracted from the nematodes at least eight times, and the extracted were mixed together. All these operations are important to improve the fidelity of the protein spots identified in this study.It is especially important for using the proteomic approach to determine the differently expressed protein spots from multiple independent experiments. Here we ran three gels for each DIGE analysis on one batch of C. elegans has only two main muscle tissues, which are the body wall for locomotion and the pharynx for feeding. The actin cytoskeleton is important for many cellular functions, including cell motility, structure and integrity. C. elegans has four actin genes that are highly homologous [Proteins contained several structural proteins, including actin (ACT-4), the myosin regulatory light chain (MLC) and tropomyosin (LEV-11), were upregulated during early hypoxic stress in the L4 stage. These proteins are typical structural proteins that are ubiquitously expressed in all eukaryotic cells and that play essential roles in myofibril assembly and muscle contraction. mologous . Isoformmologous  and aftemologous . In humamologous , howeverMlc-1 and Mlc-2 both encode a muscle regulatory myosin light chain that is involved in L1 larval viability, elongation, muscle development of the pharynx and body wall, locomotion and growth . The rolUnc-89 encodes isoform a of the muscle M-line assembly protein. UNC-89 is required for the proper organization of A bands in striated muscle and thus for normal locomotion, pharyngeal muscle contraction, and body size . UNC-89 C. elegans to maintain its shape and cell adherence. The hypoxia-induced synthesis of tropomyosin has also been reported in pulmonary arterial myocytes [C. elegans with the lev-11 gene mutant were paralyzed together with abnormal muscle filament assembly [lev-11 gene in nematode, the death rate was compared among lev-11 (x12), daf-2, daf-16 mutants and wild type worms. To the best of our knowledge, our results are the first to demonstrate that the lev-11 (x12) mutant displays a hypoxia-sensitive phenotype, which supports the idea that LEV-11 plays a role in the hypoxia response and survival in C. elegans.Interestingly, the most highly upregulated protein was tropomyosin, which was confirmed by western blot. The protein LEV-11 (spot #1) is a major component of the contractile apparatus of the muscle cells that maintain cell cytoarchitecture. LEV-11 is associated with the actin thin filament and plays a regulatory role in the organization of actin microfilaments and stress fibers. LEV-11 transmits structural changes along the thin filament during the regulatory process. The effect of increased calcium levels could be transferred to actin molecules ,48. The myocytes , Which imyocytes . C. elegassembly . To furtTherefore, we hypothesized that these cytoskeletal changes have an important role in the early hypoxia response. We propose that the synthesis of structural proteins contributes to the maintenance of the shape and function of the nematodes. These proteins are believed to be responsible for helping the worms adjust to an oxygen-deprived environment.ATP-2 encodes the beta subunit of the soluble catalytic F1 portion of ATP synthase  [The proteins ATP-2, ATP-5 and VHA-8 that were identified in this study are associated with energy and metabolism. Their altered expression levels are consistent with the altered energy metabolism seen under hypoxic conditions. mplex V) . ATP-5 e2H4+) substrate to ADP to satisfy short-term ATP requirements [C. elegans as creatine kinase does in mammalian cells [F46H5.3 encodes arginine kinase (AK), which is a member of the phosphagen (guanidino) kinase family of highly conserved enzymes that catalyze the reversible transfer of phosphate from a phosphorylated guanidine  have been reported to be induced by many types of stress. In fact, HSP70 and HSP90 family proteins are induced by hypoxia in various systems -67. The ife span . There a elegans . In thisC. elegans.After a short-term exposure to hypoxia, the protein F27D4.5 was upregulated, and the proteins ZK593.9 and Y54E5A.5 were downregulated, which was confirmed by transcript alterations. There are no clear descriptions of the molecular functions of these proteins to date. According to the GO term annotation, F27D4.5 is involved in the positive regulation of growth, and ZK593.9 is related to proteins involved in adenyl nucleotide binding, purine ribonucleotide binding, and the phosphate metabolic process. Further studies on these proteins may help understand their functions under hypoxic stress in Reactive oxygen species (ROS) have been reported to be released during hypoxia, which may contribute to the stabilization of HIF-1α ,71. Someet al. found that some metabolic enzymes, such as PYC-1, F14B4.2 hexokinase and R05F9.6 phospholglucomutase, were induced by hypoxia, which would induce the production of ATP [A comparison between hypoxia-induced mRNAs and our proteomic results was performed, but no overlap was found. The low correlation between the levels of mRNA and protein, with the activity of the protein often controlled by various post-translational modifications instead, has become widely accepted ,73. Shenn of ATP . In thisn of ATP . Therefoet al. showed that wild type C. elegans became immobile after exposure to a severe hypoxic environment (< 0.3% oxygen) but fully recovered when returned to normoxia within 4 h [C. elegans experiments were usually performed between 15-25°C. Dasgupta et al. reported that hypoxia at temperature higher than the normal physiological temperature can still induce a protective response [C. elegans at 26°C compared with a control group exposed to normoxic conditions.Scott thin 4 h . After 4thin 4 h . In mostthin 4 h ,76. Baseresponse , therefoC. elegans. The reorganization of cytoskeletal structural proteins is important for adaptation to hypoxic conditions. The preservation of the cytoskeleton and shape of cells in the presence of hypoxia would allow the worms to maintain its vital need to exchange oxygen. The synthesis of several proteins is important to maintain cell function and integrity. On the other hand, we present new evidence that translation arrest during hypoxia is induced by a downregulation of ribosomal proteins in C. elegans. This observation will help to understand the mechanisms involved in hypoxia adaptation better. Given the evolutionarily conserved functions in C. elegans (including humans), these data will broaden our understanding of how cellular remodeling might occur during hypoxia and also provide new insights into the hypoxia stress response and survival of C. elegans at the protein level. Worm cells appear to cope well during early stage hypoxia. Studies on the protein expression profiles in response to different times of hypoxia exposure will be interesting, and the resulting data obtained in C. elegans might be applicable to mammalian cells.The use of animal model systems is widely accepted as being important to understand the response to oxygen deprivation, which is involved in many human health issues, such as cancer, stroke, and cardiac failure. Given that the objective of this study was to gain a better understanding of oxygen-deprivation response and survival mechanisms, our data have provided a proteomic landscape of the molecular response to short-term hypoxia exposure in C. elegans, and this information will help broaden our knowledge on the mechanism of hypoxia response.Taken together, our data suggest that altered protein expression, structural protein remodeling, and the reduction of translation might play important roles in the early response to oxygen deprivation in C. elegans was cultured at 20°C on 9-cm nematode growth medium agar plates seeded with E. coli OP50 as described by Brenner [2PO4, 6 g/l Na2HPO4, 5 g/l NaCl, 10 mM MgSO4) from the plates, embryos were obtained by dissolving gravid animals with alkaline hypochlorite . Briefly, the pellet of worms (~0.5 ml) was resuspended and dissolved in 5 ml of an alkaline hypochlorite solution with frequent agitation. After centrifugation, the embryos that were released from the worms were washed twice with M9 buffer to remove the carcasses and then incubated at 20°C for a further 16 h to allow hatching. These worms have six development stages: embryo, L1-L4 larvae, and adult. Newly hatched L1 larvae were collected, seeded on fresh plates and incubated to the L4 larval stage. The L4 stage was selected for this study because it cannot carry embryos yet. The synchronized, cultured L4 worms were quickly harvested into a tube with M9 buffer and washed three times. The hypoxia group was incubated for 4 h in a sealed hypoxia chamber with a constant gas flow  at 26°C, and the oxygen level set to 0.2%, which was monitored by an oxygen probe. The control group was incubated for 4 h in a sealed chamber with a constant gas flow  at 26°C and the oxygen level set to normal levels (21% O2). After the 4 -h incubation, the worms were removed from the chambers and stored in liquid nitrogen before use.The wild type strain N2 (obtained from CGC) of  Brenner . After wAfter washing with M9 buffer and distilled water, the worms were resuspended in an equal volume of lysis buffer  with a protease inhibitor cocktail, frozen in liquid nitrogen, and then ground into powder with a chilled mortar and pestle. The homogenates were sonicated on ice briefly and then centrifuged at 13,000 rpm for 30 min at 4°C. The supernatants were removed and used as the protein samples. Protein samples were extracted from the nematodes at least eight times, and the extracted were mixed together. Protein concentrations were determined using the 2D Quant Kit  according to the manufacturer's protocol. The samples were stored at -80°C until use.The pH of each protein sample was adjusted to 8.5 with 50 mM NaOH, and the final concentration was adjusted to 5 mg/ml with lysis buffer. Equal amounts of protein from the sample pairs were pooled together as an internal standard. The hypoxia-treated and control samples were randomly labeled with Cy3 or Cy5, whereas the internal standards were labeled with Cy2 using 400 pmol of fluorochrome/50 μg of protein . Labeling was performed for 30 min on ice in the dark. The reactions were terminated by adding 1 μl of 10 mM lysine for 10 min on ice in the dark.After 50 μg each of the Cy3- and Cy5-labeled samples were combined, they were then mixed with 50 μg of the Cy2-labeled internal standard. An equal volume of 2× sample buffer  was added to the sample, and the total volume was increased to 410 μl with rehydration buffer . The samples were actively rehydrated into 18-cm pH 3-10 IPG strips at 17°C for 12 h using an Ettan™ IPGphor IEF System . Isoelectric focusing was performed for a total of 80 kV-h . The IPG strips were equilibrated in equilibration buffer  supplemented with 0.5% DTT for 15 min at room temperature, followed with 4.5% iodoacetamide in equilibration buffer for another 15-min incubation at room temperature.The IPG strips were placed on the top of a 12% homogeneous polyacrylamide gel that had been precast with low fluorescence glass plates using an Ettan DALT twelve gel caster. The second dimension SDS-PAGE was carried out using the Ettan DALT II . After two-dimensional electrophoresis (2DE), the gels were scanned on the Typhoon 9410 scanner with Ettan DALT gel alignment guides using excitation/emission wavelengths specific for Cy2 (488/520 nm), Cy3 (532/580 nm), and Cy5 (633/670 nm). The intensity was adjusted to ensure that the maximum volume of each image was within 60,000-90,000.t-test, and the significance level was set at P < 0.05. Protein spots that were differentially expressed in the hypoxia-treated and control groups  were marked.The gel-to-gel spot matching and statistical analyses of the protein abundances among the samples were carried out with the DeCyder 5.0 BVA  according to the manufacturer's protocol. Statistically significant differences were calculated by a paired Student's C. elegans, and we totally used three batches of C. elegans as independent experiments to meet the standard for biological replicates. Therefore, three biological replicated were included in our analyses to guarantee the statistical analysis for the samples, and the statistical values were averaged from multiple independent experiments.During our studies, we ran three gels for each DIGE analysis on one batch of For protein identification, preparative gels were loaded with 500-1000 μg of unlabeled sample. The electrophoretic conditions were the same as for the 2D-DIGE. After staining with Coomassie Brilliant Blue, the protein spots of interest were excised and destained with a solution of 25 mM ammonium bicarbonate and 50% ACN. The gels were then dried completely by centrifugal lyophilization. An in-gel digestion was performed with 0.01 μg/μl trypsin (Promega) in 25 mM ammonium bicarbonate for 15 h at 37°C. The supernatants were collected, and the tryptic peptides were extracted from the gel sequentially with 5% TFA at 40°C for 1 h and with a solution of 2.5% TFA and 50% ACN at 30°C for 1 h. The extracts were pooled and dried completely by centrifugal lyophilization.C. elegans sequences in the Swiss-Prot database using the MASCOT database search algorithms, allowing for carbamidomethylation, oxidation, and a maximum of one missed trypsin cleavage. The peptide and MS/MS tolerances were both 0.2 Da. All of the automatic data analysis and database searches were performed by the GPS Explorer™ software . Known contaminant ions (such as keratin) were excluded. A confident identification had a statistically significant (P <0.05) protein score (based on combined mass and mass/mass spectra) and a best ion score (based on mass/mass spectra).The peptide mixtures were redissolved in 0.5% TFA, and 1 μl of the peptide solution was mixed with 1 μl of matrix (4-hydroxy-α-cyanocinnamic acid in 30% ACN and 0.1% TFA) before spotting on the target plate. MALDI-TOF mass spectrometry and tandem TOF/TOF mass spectrometry were performed on a ABI-4800 Proteomics Analyzer. Peptide mass maps were acquired in positive reflection mode, averaging 1500 laser shots per MALDI-TOF spectrum and 3000 shots per TOF/TOF spectrum with a resolution of 20,000. The 4800 calibration mixtures were used to calibrate the spectrum to a mass tolerance within 0.1 Da. Parent mass peaks with a mass range of 600-4000 Da and minimum signal-to-noise ratio of 15 were selected for tandem TOF/TOF analysis. The combined mass and mass/mass spectra were used to interrogate C. elegans and control animals were separated on 12% polyacrylamide gels and transferred to PVDF membranes (Amersham Biosciences). These blots were incubated for 2 h at room temperature in Tris-buffered-saline with Tween  containing 5% skim milk. The blots were incubated with an anti-tropomyosin monoclonal antibody  overnight at 4°C. After washing three times in Tris-buffered-saline with Tween, the blots were incubated with a horseradish peroxidase-conjugated secondary antibody  for 1 h at room temperature. Immunoreactive complexes were visualized using ECL reagents . The housekeeping protein α-tubulin was used as an internal control . Protein expression levels were quantified with the ImageJ software. The data was statistically analyzed by OriginPro v8.0 software using a paired Student's t-test, with P <0.05 indicating a significant difference.Proteins from the hypoxia-treated lev-11, daf-2 (hypoxia-resistant), daf-16 (hypoxia-sensitive) mutant strains were kindly provided by the CGC and cultured as described above. Approximately 100 to 150 L4 larvae from each of the three mutant strains and the wild type (N2) were quickly harvested in a tube with M9 buffer, washed three times, and incubated for 8 h, 10 h,12 h, 14 h and 16 h in a sealed hypoxia chamber . The worms were then cultured at normoxia  on 3-cm nematode growth medium agar plates seeded with E. coli OP50. After 24 h, the numbers of living and dead worms were counted, which were used to calculate the death rate. Worms without spontaneous or touch-evoked movement were scored as dead. The experiments were repeated at least three times (n > 3). The statistical analysis was performed by OriginPro v8.0 software using a paired Student's t-test, with P <0.05 indicating a significant difference.The http://david.abcc.ncifcrf.gov/.Searches for the gene ontology (GO) classifications of the proteins identified by MALDI/TOF/TOF were performed using the web-accessible DAVID annotation system The authors declare that they have no competing interests.C. elegans models for this study and completed the additional studies for revising the manuscript. XH helped on the bioinformatic analysis. FZ, YG and YW helped on the Western blot analysis. LX and CC gave English writing suggestions and statistical analyses on this work. CZ designed and supervised the study, finalized the manuscript. All authors read and approved the manuscript.HL wrote the main manuscript and did the most of the experiments. CR and JS helped on the


We examined changes in the ultrastructure and localization of major extracellular matrix components, including 5 types of collagen , laminin, fibronectin, and heparan sulfate proteoglycan in Descemet's membrane of the cornea of diabetic GK rats. In the cornea of diabetic GK rats, more long-spacing collagen fibrils were observed in Descemet's membrane than in the membrane of the nondiabetic Wistar rats. Both GK and Wistar rats showed an age-dependent increase in the density of the long-spacing collagen. Immunoelectron microscopy showed that type VIII collagen was localized in the internodal region of the long-spacing collagen, which was not labelled by any of the other antibodies used. The antidiabetic agents nateglinide and glibenclamide significantly suppressed the formation of the long-spacing collagen in the diabetic rats. Long-spacing collagen would thus be a useful indicator for studying diabetic changes in the cornea and the effect of antidiabetic agents. Corneal keratopathy is one of thediabetic complications. Clinically, the diabetic cornea often shows superficialpunctate keratopathy and persistent epithelial defects and recurrent epithelialerosion that are considered to be a form of diabetic keratoepitheliopathy.Various degrees of epithelial disturbance take place in the diabetic cornea.Thickening of epithelial basement membrane in the diabetic cornea has beenextensively studied , 2.In aThe diabeticcorneal endothelium has been shown by speculum-aided microscopy to have morphologicalabnormalities such as polymorphism . The endThe spontaneouslydiabetic Goto-Kakizaki (GK) rat is a nonobese model of type 2 diabetes that wasdeveloped by the selective breeding of glucose-intolerant Wistar rats –15. In tn = 6 for each age) Goto-Kakizaki rats and Wistar rats ,obtained from Kurea , wereused in the present study. Rats werehoused under 12-hour light: 12-hour dark cycle and given free access to food and water. Serumglucose levels in Wistar and GK rats, which were measured after an overnightfast, were, respectively, 158.0 ± 12.0 and 375.9 ± 11.6 (mean ± SEM) mg/dL at 15 weeks, 118.5 ± 10.5 and 333.8 ± 22.4 mg/dL at 33 weeks, and167.0 ± 28.6 and 314.9 ± 48.7 mg/dL at 62 weeks. Asreported previously [All experimental procedures usinglaboratory animals were approved by the Animal Care and Use Committee of KyorinUniversity School of Medicine. The corneas of 15-, 33-, and 62-week-old male against Type VIII collagen and a monoclonal antibody (clone 15B6) againstType VI collagen were prepared and characterized as described previously , 20. Polμm thick) were made, washed with PBS, and incubated for 10minutes in 5% BSA in PBS. The sections were then incubated withanti-extra-cellular matrix component antibody for 1 hour at room temperature,washed with PBS, and subsequently incubated with Alexa 568-conjugated donkeyanti-rabbit or mouse IgG antibody (1:200). Nuclei were stained with SYBR-GreenI (1:500). After a final wash with PBS, the specimens were mounted in 90%glycerol-0.1 M Tris-HCl buffer (pH8.5) containing 0.5 mM p-phenylene diamine,and observed under a laser scanning confocal microscope . For a controlexperiment, the specimens were incubated with normal rabbit or mouse IgG orwith 0.1% BSA-PBS alone instead of the primary antibodies. No positive stainingwas observed in the control experiment (data not shown).Immunofluorescenceobservation for localization of components of extracellular matrix wasperformed as described earlier . CorneassO4 in 0.1 M phosphate buffer (pH7.4), and dehydrated with gradedalcohols. After immersion in propyleneoxide, the specimens were embedded in Epon 812. Ultrathin sections were cut perpendicular to the epithelium, doublystained with uranyl acetate and lead citrate, and examined with a transmissionelectron microscope, TEM-1010 .Corneas were fixed inphosphate-buffered 2.5% glutaraldehyde (pH7.4). Strips of cornea were takenfrom the central part of the cornea, and were postfixed in 1% OImmunoelectron microscopicobservation for localization of components of extracellular matrix wasperformed as described earlier . FixatioThe administration of antidiabetic drugswas started at 8 weeks old. Nateglinide (50 mg/kg) or glibenclamide (2 mg/kg)was suspended in 0.5% methylcellulose and administered to GK rats via a stomachtube in volume of 10 mL/kg –25. Thest-test. Differences were defined as significant at P < .05.Results were expressed as the mean ± standard deviation, and the statistical analysis was done by the use of the unpairedStudent's μm innormal rats and 0.5–2.15 μm in the diabetic ones. In the diabetic cornea, thenumber of long-spacing collagen fibrils in the membrane increased more than inthe normal cornea with aging. Thedensity of these collagen fibrils was higher adjacent to the endothelium andlower toward the stroma  were frequently observed in Descemet'smembrane of diabetic rats, whereas they were observed less so in that of thenormal rats. e stroma .It was earlier shown that type VIIIcollagen is localized in Descemet's membrane . So weeLocalization of type VIII collagen inDescemet's membrane was examined immunoelectron-microscopically by using thecolloidal-gold labeling method . In theNext we examined the localization of theother components of the extracellular matrix, that is, laminin and type I, III,IV, and VI collagens. There is a report that type VI collagen becomes localizedin the long-spacing collagen in the corneoscleral meshwork of human eyes . So we eP < .05) inhibited the formation of the long-spacing collagen inthe diabetic GK rats (The morphological change in long-spacingcollagen is thought to be one of the complications of diabetes. Thus weexamined whether the abnormal formation of long-spacing collagen could be suppressedby the antidiabetic agents nateglinide and glibenclamide. As shown in ourprevious study , during  GK rats . Glibenc GK rats .Ourpresent study revealed that a diabetes-associated increase in the number oflong-spacing collagen fibrils occurred in Descemet's membrane in the cornea oftype II diabetes model GK rats. This result is consistent with the findingsmade in the corneas of diabetic humans and type I diabetes model Streptozotocin-induceddiabetic rats , 12.TheImmunoelectronmicroscopy showed that the long-spacing collagen contained type VIII collagenmolecules . HoweverLong-spacingcollagen is also observed in the eyes from patients withiridocorneal-endothelial syndrome, primary open-angle glaucoma, age-relatedmacular degeneration, and Fuchs' endothelial corneal dystrophy , 32–34. Diabetesinduces the dysfunction of corneal endothelium –42. HighD-Phenylalanine derivative drug nateglinide and sulfonylurea drugglibenclamide are antidiabetic agents that increase insulin secretion. When weexamined the effect of nateglinide and glibenclamide on the morphologicalchanges in Descemet's membrane of GK rats, we found that the abnormal formationof the long-spacing collagen was significantly suppressed by the administrationof either nateglinide or glibenclamide . These rIn the present study, glibenclamide treatment was more effectivethan nategelinide treatment . The difInsummary, more long-spacing collagen fibrils were observed in Descemet'smembrane of diabetic GK rats than in the membrane of the nondiabetic Wistarrats. Type VIII collagen was localizedin the internodal region of the long-spacing collagen. Further studies are needed to elucidate therole of type VIII collagen in the formation of long-spacing collagen. Antidiabeticagents nateglinide and glibenclamide significantly suppressed the formation ofthe long-spacing collagen in the diabetic rats. Thelong-spacing collagen of the cornea would appear to be a useful indicator forstudying diabetic changes in the cornea and the effect of antidiabetic agents.


Neuroendocrine deficiencies may affect recovery after aneurysmal subarachnoid hemorrhage (aSAH). Insulin like growth factor-I (IGF-I) regulates neuronal growth and apoptosis in ischemic stroke. Our study was designed to a) characterize the behavior of serum IGF-I and growth hormone (GH) in the acute and late phases after aSAH reflecting possible pituitary gland function and b) evaluate the association between IGF-I and morbidity assessed by Glasgow outcome scale (GOS) and health related quality of life (HRQoL) in patients with aSAH.In this prospective cohort study, patients with aSAH (n = 30) were compared to patients who underwent elective aneurysm surgery (n = 16). Serum GH and IGF-I concentrations were measured daily for five (controls) or seven (aSAH) days and at three months. GOS and 15d HRQoL was measured at three months. A mixed models method was used for testing between the groups. For factors possibly affecting HRQoL in aSAH patients, we constructed a Bayesian predicting model using a P-course Bayesian classifier.P = 0.01). No corresponding difference was found at three months. Serum GH concentrations were similar in both patient groups. Severity of the aSAH did not affect serum IGF-I concentrations. Patients with GOS ≤ 4 had lower IGF-I concentrations and lower HRQoL than patients with GOS 5 (P = 0.02 and 0.003 respectively). The 15d HRQoL was 0.81 ± 0.16 in patients with aSAH and 0.86 ± 0.09 in control patients (P = 0.24). In the Bayesian model, the use of statins prior to aSAH, hyponatremia, high maximal sequential organ specific score (SOFAmax), and low cumulative IGF-I concentrations on days one to seven were associated with poor HRQoL .The mean IGF-I concentrations for days one to five were 8.1 ± 3.5 nmol/l in patients with aSAH and 11.2 ± 3.1 in the control group (IGF-I concentrations are low during acute aSAH, which may have an impact on morbidity.ClinicalTrials.gov Identifier NCT00614887 Acute aneurysmal subarachnoid hemorrhage (aSAH) is a devastating disease, annually affecting 9 in 100,000 people .Some studies suggest the hypothalamo-pituitary-adrenal (HPA) axis may already be affected in the acute phase of aSAH -6. SeverThere is no data available on the behavior of serum IGF-I concentrations in the acute phase of aSAH. The aim of our study was to characterize the behavior of IGF-I and GH acutely and three months after aSAH. This may reflect acute pituitary function. Additionally, we hypothesized that low cumulative IGF-I concentrations may negatively influence morbidity assessed by Glasgow outcome scale (GOS) and health-related quality of life (HRQoL).All patients aged 18 years or over and scheduled for an elective open surgical aneurysm treatment (control group), and all patients with aSAH admitted to Kuopio University Hospital in Finland between 29 March and 30 November 30, 2006 were prospectively assessed for eligibility for this study. Patients with an elective endovascular management of their unruptured aneurysm were excluded from the control group because of their expectedly short hospital stay. This study's patient population is the same as that used in a previous publication [The hospital ethics committee approved the study protocol and informed written consent was received from the patients or their next of kin.The following blood samples were collected from the first to seventh mornings following bleeding: serum (s) GH , serum IGF-I . In control patients, the corresponding blood samples were collected from the first to fifth postoperative days after the patients were discharged. Additional routine laboratory parameters, such as electrolytes, were collected on a daily basis.At the scheduled three-month follow-up visit, serum GH and serum IGF-I samples were collected at 9 am. In addition, the patients (or their relatives) were asked to fill out a 15D quality of life questionnaire . We usedThe HRQoL was measured by the 15D scale ,20. The We used a power of 80% and a two-sided α-level of 0.05 in sample size calculations. We assumed that 25% of patients with aSAH and none of the elective surgical patients would develop pituitary insufficiency measured by low IGF-I. Data are presented as mean ± standard deviation, absolute values and percentages, or medians and interquartile ranges. Distribution of the data was assessed by the Kolmogorov-Smirnov test. For normally distributed parameters, student's t-tests were used to compare the means of different groups. The Mann-Whitney U test was used for nonparametric testing between the groups. A mixed models method was used to test between groups, allowing heterogeneity between the groups. To identify the factors associated with poor HRQoL or death, we used a Bayesian predicting model in aSAH patients. This was performed using P-course Bayesian classifier .a priori information.P-course is a web-based Bayesian classifier that is able to use multidimensional priors, for example separate priors for the outcome variable, in general, and for the outcome variable according to each predicting variable. The methods have equaled or outperformed novel logistic regression, especially in small data sets, in terms of prediction accuracy , variablThe outcome variable was poor HRQoL measured by 15D and dichotomized into normal (0.80 to 1.00) or poor (0 to 0.79), where the value 0 indicated death. In the first phase, there were 355 potential predicting variables. By using P-course classifier, this was reduced to 22 variables from 30 aSAH patients. To avoid over-fitting the model, we formed four randomly selected sets of 25 patients, and a prediction model was performed for each set. We obtained four slightly different sets of prediction variables.We recruited 30 patients with aSAH and 16 control patients who underwent elective aneurysm surgery. We were unable to recruit the planned 30 control patients because endovascular aneurysm treatment was frequently chosen [P < 0.001). The three-month mortality was 10% in patients with aSAH. None of the patients in the control group died.Seventeen patients with aSAH underwent endovascular treatment and 13 patients had open surgery. The mean length of stay (LOS) was 97 ± 100 hours at the ICU and 34 ± 51 hours at the high-dependency unit (HDU) in patients with aSAH. Patients in the control group had an HDU LOS of 22 ± 2 hours. Patients with aSAH had a hospital LOS of 14.6 ± 5.4 days, and patients in the control group of 8 ± 3 days (P = 0.01), but no difference was found at three months follow up .In patients with aSAH, there were no differences in IGF-I or GH concentrations between the patients with respect to aneurysm location (anterior communicating artery versus others), treatment modality (clip vs. coil), Hunt-Hess grades (I to III versus IV to V), Fisher grade (I to II versus III to IV), Glasgow Coma Scale (GCS) (less than 8 or more than 8), symptomatic vasospasm and/or need for norepinephrine (n = 9), or hydrocephalus on admission. Gender or body mass index did not affect IGF-I or GH concentrations. Age was negatively correlated with IGF-I . Quality of life was moderately low (<0.8) in six patients with aSAH and in five control patients (P = 0.75). In addition, three patients with aSAH and none of the control patients had poor quality of life (<0.6). The HRQoL dimensions of speech (P = 0.04) and eating (P = 0.03) were lower in patients with aSAH than in the control group; otherwise, no statistically significant differences were observed in the scores between the groups. Ten patients in the aSAH group had a GOS of four or less, and the others had a GOS of five. Patients with a GOS of four or less had lower quality of life than patients with a GOS of five . Additionally, patients with a GOS of four or less had lower mean IGF-I concentrations than patients with a GOS of five . Patients with Hunt & Hess grades IV to V had similar HRQoL than patients with Hunt & Hess grades I to III . aSAH patients with low HRQoL (<0.8) had lower mean IGF-I concentrations than patients with good HRQoL (5.7 ± 2.1 nmol/l vs. 8.7 ± 3.4 nmol/l). Mean IGF-I for days one to seven was mildly associated with HRQoL by a Pearson's correlation coefficient of 0.36 (P = 0.08).The mean 15D HRQoL sum in patients with aSAH was 0.81 ± 0.16 and in control patients 0.86 ± 0.09 , log-score 0.69). When the model was tested by putting the total material into the model, an accuracy of 89% was reached. The sensitivity was 0.86 and specificity was 0.93. The results indicate that a low IGF level in the days following aSAH may be associated with poor HRQoL or death.To the best of our knowledge, this is the first study to evaluate the behavior of IGF-I in the acute phase of aSAH, and to combine it with GOS and HRQoL assessment at three months. According to our study, serum IGF-I levels are low during acute aSAH, but they normalize at three months. The results may reflect diminished acute pituitary function. The severity of aSAH does not influence serum IGF-I levels. Quality of life at three months was equal in patients with acute alive aSAH at three months and patients who underwent elective surgery for unruptured aneurysms. However, low IGF-I values, measured acutely after aSAH, may predict morbidity assessed by GOS and HRQoL.aSAH may cause various long-lasting neurological deficits and disturbances in mental health, sleep, concentration capability, anxiety, and depression . These sIn the context of aSAH, it is most interesting that IGF-I presents with major vascular effects, and thus may contribute to the pathophysiological processes in aSAH .per se [Serum IGF-I is essential in mediating GH action. During critical illness, serum levels of IGF-I may decrease substantially . There iper se .Low IGF-I concentrations in aSAH may reflect either pituitary GH insufficiency, decreased liver production, or a stress-related decrease of IGF-I concentrations in critical illness. An essential percentage of aSAH patients had IGF concentrations far below the age-related reference values. It was shown previously that a disturbance in the function of the GH-IGF axis is the first pituitary deficiency to appear after aSAH or TBI ,29. The The role of single IGF-I concentration measurement may be controversial. It is generally accepted that definitive GH insufficiency should be tested by means of a stimulation test . ConcentIn our study, GH concentrations and IGF-I concentrations did not correlate with each other. GH concentrations vary a lot during critical illness; the pulsatile secretion of GH makes adequate interpretation of serum concentrations of GH especially difficult. In the present study, GH concentrations were equal in patients with aSAH and the control patients. In the literature, there are no data on GH concentrations in the acute phase of aSAH. At the later phase, after aSAH, GH insufficiency may appear in 25% of patients .There are some limitations in our study. We did not use the GH stimulation test when diagnosing GH deficiency. However, as described above, IGF-I itself has an essential role in acute neurological diseases ,12. The We propose that it might also be argued that the method of securing the ruptured aneurysm in the aSAH group could markedly effect the results. However, severity of the bleeding and the level of consciousness on admission are by far the most important prognostic factors in aSAH and the treatment modality is of lesser importance ,34. As eIGF-I may be one factor among multiple others in influencing outcome in patients with aSAH. Larger studies are needed to prove the causality of this finding.Serum IGF-I concentrations are low in patients with acute aSAH, which may affect morbidity in these patients. The severity of aSAH did not affect IGF-I concentrations. Larger studies are needed to evaluate the neuroprotective role of IGF-I in aSAH.• IGF-I has an important role in neuronal growth and cell death• Serum IGF-I concentrations are low in the acute phase of aSAH• Low IGF-I concentrations may reflect acute pituitary insufficiency in patients with aSAH• Low IGF-I concentrations may affect morbidity in patients with aSAHaSAH: aneurysmal subarachnoid hemorrhage; ELISA: enzyme-linked immunosorbent assay; GH: growth hormone; GCS: Glasgow Coma Scale; GOS: Glasgow outcome scale; HDU: high-dependency unit; HPA: hypothalamo-pituitary-adrenal; HRQoL: health-related quality of life; IGF-I: insulin-like growth factor-I; LOS: length of stay; TBI: traumatic brain injury.The authors declare that they have no competing interests.SB, AU and TK participated in study conception, acquisition of data, analysis and interpretation of data, drafting the manuscript and revising it critically. OR participated in analysis and interpretation of data, drafting the manuscript and revising it critically. ER, JRO and VK participated in analysis and interpretation of data.


AVI-014 is an egg white-derived, recombinant, human granulocyte colony-stimulating factor (G-CSF). This healthy volunteer study is the first human investigation of AVI-014.24 male and female subjects received a single subcutaneous injection of AVI-014 at 4 or 8 mcg/kg. 16 control subjects received 4 or 8 mcg/kg of filgrastim  in a partially blinded, parallel fashion.The Geometric Mean Ratio (GMR) (90% CI) of 4 mcg/kg AVI-014/filgrastim AUC(0–72 hr) was 1.00  and Cmax was 0.86 . At the 8 mcg/kg dose, the AUC(0–72) GMR was 0.89  and Cmax was 0.76 . A priori pharmacokinetic bioequivalence was defined as the 90% CI of the GMR bounded by 0.8–1.25. Both the white blood cell and absolute neutrophil count area under the % increase curve AUC(0–9 days) and Cmax GMR at 4 and 8 mcg/kg fell within the 0.5–2.0 a priori bound set for pharmacodynamic bioequivalence. The CD 34+ % increase curve AUC(0–9 days) and Cmax GMR for both doses was ~1, but 90% confidence intervals were large due to inherent variance, and this measure did not meet pharmacodynamic bioequivalence. AVI-014 demonstrated a side effect profile similar to that of filgrastim.AVI-014 has safety, pharmacokinetic, and pharmacodynamic properties comparable to filgrastim at an equal dose in healthy volunteers. These findings support further investigation in AVI-014. Granulocyte colony stimulating factor (G-CSF) is a cytokine produced by monocytes, macrophages, endothelial cells, and fibroblasts. Human G-CSF consists of 174 amino acids with an approximate molecular weight of 20 kDa. The native protein is O-glycosylated on threonine 133. G-CSF plays a critical role in the modulation of neutrophil biology. It is required to maintain adequate basal neutrophil count, as well as generation of an appropriate neutrophilia in response to infectious stimuli [E. coli bacteria transfected with rhG-CSF cDNA. Two other products approved in several countries include lenograstim, which is derived from mammalian cells and is glycosylated, and nartograstim which is non-glycosylated and has 5 different amino acids at the N-terminal region as compared with the natural human G-CSF. All the three versions of rhG-CSF have been successfully used to treat chemotherapy-induced or other forms of neutropenia for over a decade and appear to have similar efficacy [Filgrastim is a recombinant human granulocyte colony stimulating factor (rhG-CSF) which was developed in the mid-1980's and FDA-approved for use in chemotherapy induced neutropenia in 1991 . Filgrasefficacy . Specifi8 IU/mg) in NFS-60 cells shows equipotency to filgrastim.AVI-014 is a transgenic glycosylated rhG-CSF produced by purification from egg whites of hens transfected with hG-CSF cDNA. The threonine residue at position 133 is glycosylated and the protein has a molecular weight of approximately 20 kD. Matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis shows this species has the same polypeptide sequence as the natural human G-CSF. AVI-014 shows immunoreactivity with rhG-CSF-specific antibodies in a Western blot assay and bioactivity in a cell proliferation assay. Biologic activity . No subject used prescription or over-the-counter medications, with the exception of hormonal contraceptives, within the period starting 14 days prior to first administration of the study drug. Allergy to chicken eggs or influenza vaccine was an exclusion criteria. Subjects with a body mass index between 19 and 30 were enrolledThis was a single escalating dose study comprised of three treatment groups. Eight subjects in Panel A received an open label, single subcutaneous dose of AVI-014 at a dose of 4 μg/kg. Subjects in Panel B were administered with either a single subcutaneous dose of AVI-014 4 μg/kg (n = 8) or an equivalent dose of filgrastim  4 μg/kg (n = 8) in a double blinded fashion. Subjects in Panel C were administered with either a single subcutaneous dose of AVI-014 8 μg/kg (n = 8) or an equivalent dose of filgrastim 8 μg/kg (n = 8) in a double blinded fashion. This was a first-in-human administration of AVI-014, and no formal power calculation was performed. Prior to unblinding, the computer-generated randomization scheme was accessible only to the study statistician and to the unblinded study staff member. Neither of these individuals assessed adverse events. All pharmacokinetic and pharmacodynamic analysis was performed prior to unblinding.Each subject received a comprehensive medical history and inquiry into concomitant medication use, complete physical examination, clinical laboratory safety assessments , and ECG during screening. Females had a negative serum pregnancy test during screening as well as a negative urine pregnancy test on the first day of the study prior to study drug administration. Subjects received a single subcutaneous injection in the abdomen of study drug followed by pharmacokinetic and pharmacodynamic sampling. ECG examination was performed at screening, within 1 hour prior to dosing, and at 6 hours and 21 days post study drug administration. Injection sites were monitored for signs of irritation or inflammation.Blood for pharmacokinetics was drawn at baseline and 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 24, 48, and 72 hours after drug administration. Blood samples were allowed to clot and serum was collected and stored at <-20°C. Serum levels of AVI-014 and control filgrastim were determined using a hG-CSF specific ELISA kit . Concentration was calculated by interpolation using a standard curve generated with either AVI-014 or filgrastim. The limit of quantification of the assay was 7.8 pg/ml.Pharmacodynamic endpoints included absolute neutrophil count (ANC), total white blood cell (WBC) count and total CD 34+ cell count. A complete blood count was obtained predose and on days 2–9. Blood for flow cytometry was collected predose and days 2–6. With the exception of the predose draw, which was collected at approximately 20:00 on day -1, blood for pharmacodynamic evaluation was collected at the dose time. CD 34+ counts were performed by the Thomas Jefferson University Hospital Clinical Laboratory. During flow cytometry of subject samples, the gated area for CD 34 positive cells was determined by visual inspection with an effort to maintain consistency between samples. All flow cytometry and clinical laboratory personnel were blinded during the conduct of the study.Pharmacokinetic analysis was performed using WinNonLin Enterprise version 5.1 . A non-compartmental approach was employed. Area under the curve (AUC) was determined using the linear/log trapezoidal rule. Actual sample collection times were used for the generation of pharmacokinetic parameters. Individual time concentration plots were examined for each subject. Appropriateness of time points to be used in calculation of the elimination rate constant was verified by visual examination of the terminal time points. In all cases the terminal elimination was determined from the 24 to 72 hour time point. Estimation of an alpha half life was performed using the time points of 8 to 24 hours.AUC's and Cmax values were natural log-transformed. Means, standard deviations, min, max and medians were calculated for each dose level and group (AVI-014 and filgrastim). Arithmetic means were be back-transformed (by taking anti-logs) to obtain geometric means. The a priori bounds that would define pharmacokinetic equivalence of AVI-014 and filgrastim were a 90% confidence interval of geometric mean ratio of area under the time concentration curve (AUC(0–72)) and of the maximal G-CSF concentration (Cmax) that lies between 0.8 and 1.25. A linear mixed effects model was used to compare AVI-014 and filgrastim for the parameters of AUC(0–72) and Cmax. This model had treatment as a fixed effect. Panel and session nested within panel were also fixed effects. The variances were calculated for each group, using subject as random effects. Ratios (AVI-014/filgrastim) of geometric means were calculated by back-transforming the difference of least-squares mean, and a 90% confidence interval calculated for this ratio. Residuals were computed and examined for normality.WBC, ANC and CD 34+ were analyzed as percentage increase from the baseline, with determination of an area under the % increase curve (AUC(0–9 day) for WBC and ANC, and AUC(0–6 day) for CD34+), the maximal % increase from baseline (Cmax). Units for AUC are % increase-day; and for Cmax are % increase. The distance between day 0 and day 2 was considered to be 1.5 days for the purposes of determination of area under the curve. Geometric mean ratios of AUC and Cmax were compared, and the data were analyzed in the same fixed effects model as for pharmacokinetics. The a priori bounds that defined pharmacodynamic equivalence of AVI-014 and filgrastim were a 90% confidence interval of geometric mean ratio of area under the % increase curve (AUC) and of the maximal % increase from baseline (Cmax) that lay between 0.5 and 2.0.The mean age of the subjects enrolled in the trial was 34.5 years range 21–50). Twenty nine (73%) of the subjects were male. Thirteen (32.5%) of the subjects were white and 27 (67.5%) were black. The mean weight was 81 kg (range 61–106). Demographics by treatment allocation are presented in Table –50. TwenA summary of PK parameters by treatment for all groups is presented in Table Peak WBC and ANC occurred on day 2, with a return to baseline values at approximately day 6. CD 34+ peak values occurred on day 3 for the 4 μg/kg groups and day 4 for the 8 μg/kg groups. AVI-014 and filgrastim demonstrate similarity in WBC, ANC, and CD 34+ response at equal mass doses of drug. Geometric mean ratios of ANC, WBC and CD 34+ cell counts between treatments are presented in Tables All subjects completed the trial. There were no serious adverse events. A total of 47.5% of the enrolled subjects experienced at least one adverse event. Of a total of 42 adverse events, thirty-four events were judged as possibly related to study drug. The most common adverse events were headache, myalgia, back pain, and bone pain. 50.0% (12 of 24) of subjects treated with AVI-014 and 43.8% (7 of 16) of those treated with filgrastim had an adverse event. Musculoskeletal complaints, which included bone pain and muscle aches, occurred in 37.5% (9 of 24) subjects receiving AVI-014 and 18.8% (3 of 16) subjects receiving filgrastim. Headache occurred in 20.8% (5 of 24) subjects receiving AVI-014 and 18.8% (3 of 16) of subjects receiving filgrastim. A sensation of neck fullness was noted in two subjects on the day of drug administration. One subject noted headache, dizziness, chest discomfort, and nausea approximately 45 minutes after receiving AVI-014 4 μg/kg. This resolved and approximately eight hours later this subject noted a sensation of neck fullness. The event in this subject occurred 30 minutes after his roommate, who received filgrastim 4 μg/kg, noted neck fullness. Both episodes were self-limited, not associated with other signs of hypersensitivity, and considered possibly related to study drug. Injection site pain occurred in one subject receiving AVI-014.For all subjects, serum neutralizing antibodies and binding antibodies were assessed by serum-mediated inhibition of in vitro cell proliferation assays and by ELISA detection/quantification of hα-G-CSF antibodies using goat α-human Ig-peroxidase conjugate (ZeptoMetrix). Blood sampling for immunogenicity testing was performed at pre-dose and on day 21 time points. No hG-CSF neutralizing antibodies were detected in pre-dose or day 21 samples from any subject. One patient who had received AVI-014 at 8 μg/ml showed low levels (<3 ng/ml) of anti-hG-CSF antibodies in both pre-dose and day 21 samples, with equivalent levels at both time points. Low levels of naturally occurring anti-G-CSF antibodies in normal subjects have previously been reported to be relatively common (11% of subjects) . Thus, nThis study describes the first human administration of AVI-014, an avian-derived, human, glycosylated G-CSF. AVI-014 was well tolerated, with a side effect profile of myalgia and bone pain that was comparable to that seen with a similar dose of filgrastim. The product insert for filgrastim notes a ~3.5 hour elimination half life ,8, whichThe comparative extent of G-CSF exposure, as determined by AVI-014 and filgrastim AUC(0–72) values, appeared comparable at 4 μg/kg and 8 μg/kg dose. However, the 90% confidence interval of the geometric mean ratio is outside a priori bioequivalence bounds of 0.8–1.25. It should be noted that this study employed a parallel and not crossover design and thus does not constitute a formal exploration of bioequivalence. A parallel design was chosen because of unknown immunogenicity of AVI-014 in this first-in-human clinical trial. There was no evidence of immunogenicity noted in this trial, which is consistent with the experience of an egg white-derived interferon alpha-2b . The useThe response of each product in terms of WBC, neutrophil response and CD 34+ was similar between treatments. This is consistent with previous observations which demonstrated a close linkage between the serum concentration of recombinant G-CSF and neutrophil response , as wellIn conclusion, AVI-014 was well tolerated in healthy subjects with no evidence of immunogenicity. AVI-014 has a pharmacokinetic profile in healthy volunteers that is comparable to that of filgrastim and generates a pharmacodynamic response in terms of neutrophil mobilization that is equivalent to that of filgrastim. The present report supports further investigations of AVI-014 in patient populations.CI: confidence interval; ECG: electrocardiogram; G-CSF: granulocyte colony-stimulating factor; rhG-CSF: recombinant human granulocyte colony stimulating factor; GMR: Geometric Mean RatioThis study was funded by Synageva Biopharma . Dr. Varki was supported by NIH Postdoctoral training grant T32 GM08562. Dr. Kraft has received research grants from, and is a paid consultant to, Synageva Biopharma.RV prepared the first draft of the manuscript, edited revisions, and assisted in the conduct of the clinical trial. EP performed statistical analysis. MCL performed analytical analysis, edited the manuscript drafts and revisions, and contributed to the study design. AF assisted with study design and conduct of the clinical trial. WKK designed study protocol, performed pharmacokinetic analysis, revised and edited first draft of manuscript, prepared revised drafts of the manuscript, and was responsible for overall conduct of the clinical trial. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


RNA interference (RNAi), mediated by 21-nucleotide (nt)-length small interfering RNAs (siRNAs), is a powerful tool not only for studying gene function but also for therapeutic applications. RNAi, requiring perfect complementarity between the siRNA guide strand and the target mRNA, was believed to be extremely specific. However, a recent growing body of evidence has suggested that siRNA could down-regulate unintended genes whose transcripts possess complementarity to the 7-nt siRNA seed region. This off-target gene silencing may often provide incongruous results obtained from knockdown experiments, leading to misinterpretation. Thus, an efficient algorithm for designing functional siRNAs with minimal off-target effect based on the mechanistic features is considered of value.We present siDirect 2.0, an update of our web-based software siDirect, which provides functional and off-target minimized siRNA design for mammalian RNAi. The previous version of our software designed functional siRNAs by considering the relationship between siRNA sequence and RNAi activity, and provided them along with the enumeration of potential off-target gene candidates by using a fast and sensitive homology search algorithm. In the new version, the siRNA design algorithm is extensively updated to eliminate off-target effects by reflecting our recent finding that the capability of siRNA to induce off-target effect is highly correlated to the thermodynamic stability, or the melting temperature (Tm), of the seed-target duplex, which is formed between the nucleotides positioned at 2-8 from the 5' end of the siRNA guide strand and its target mRNA. Selection of siRNAs with lower seed-target duplex stabilities (benchmark Tm < 21.5°C) followed by the elimination of unrelated transcripts with nearly perfect match should minimize the off-target effects.http://siDirect2.RNAi.jp/.siDirect 2.0 provides functional, target-specific siRNA design with the updated algorithm which significantly reduces off-target silencing. When the candidate functional siRNAs could form seed-target duplexes with Tm values below 21.5°C, and their 19-nt regions spanning positions 2-20 of both strands have at least two mismatches to any other non-targeted transcripts, siDirect 2.0 can design at least one qualified siRNA for >94% of human mRNA sequences in RefSeq. siDirect 2.0 is available at  Archaeoglobus fulgidus Ago-like protein , where ΔH  is the sum of the nearest neighbor enthalpy change, A is the helix initiation constant (-10.8), ΔS is the sum of the nearest neighbor entropy change  was fixed at 100 mM. As shown in our previous report, calculated Tm of 21.5°C may be a benchmark to discriminate almost off-target-free seed sequences from the off-target-positive ones [We have found that the off-target effect is highly correlated with the thermodynamic stability or Tm of the seed-target duplex, which is formed between the nucleotides positioned at 2-8 from the 5' end of the siRNA guide strand and its target sequence . In the ive ones , and thuive ones , and thaive ones ,28. ThusSeveral studies have indicated that the effect of single-base mismatches between the siRNA guide strand and the target mRNA varies, according to the positions of the mismatch and/or the sequence of siRNA ,29. HoweWe performed a genome-wide design of siRNAs for human mRNAs in RefSeq release 30 with the following parameters: 1) satisfying our functional siRNA design algorithm ,24, 2) TThe degree of off-target effects is shown to be correlated with the thermodynamic stability or the calculated Tm value of the seed-target duplex . The iniIt is also desirable to select siRNA that contains as many mismatches as possible to any non-targeted mRNAs. In addition to the Tm value of below 21.5°C, siRNA sequences with at least two mismatches to any other non-targeted transcripts are selectable for 94.7% of human mRNAs Figure . HoweverWe were unable to design functional, off-target minimized siRNAs for 5.3% of the RefSeq mRNAs using the default parameters. Typical examples of these mRNAs are the histone clusters  and ribosomal proteins , which are known to form multigene families. When designing siRNAs targeting such genes, users can manually investigate the detailed list of off-target gene candidates Figure  and sele7 = 16,384) ranged from -12°C to 60°C, and of these, 4488 (27.4%) 7-mers had the Tm below 21.5°C  functional siRNAs that satisfy our guideline , 2) siRNProject name: siDirecthttp://siDirect2.RNAi.jp/Project home page: Operating system(s): Platform independentProgramming language: Perllicense@RNAi.jpAny restrictions to use by non-academics: Contact YN developed the webserver and performed the computational analyses. JY and SM developed the core framework of the software. KU-T supervised the entire study. YN and KU-T drafted the manuscript. All authors read and approved the final manuscript.


Neurotransmitter release at CNS synapses occurs via both action potential-dependent and independent mechanisms, and it has generally been accepted that these two forms of release are regulated in parallel. We examined the effects of activation of group III metabotropic glutamate receptors (mGluRs) on stimulus-evoked and spontaneous glutamate release onto entorhinal cortical neurones in rats, and found a differential regulation of action potential-dependent and independent forms of release. Activation of presynaptic mGluRs depressed the amplitude of stimulus-evoked excitatory postsynaptic currents, but concurrently enhanced the frequency of spontaneous excitatory currents. Moreover, these differential effects on glutamate release were mediated by pharmacologically separable mechanisms. Application of the specific activator of adenylyl cyclase, forskolin, mimicked the effect of mGluR activation on spontaneous, but not evoked release, and inhibition of adenylyl cyclase with 9-tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536) blocked mGluR-mediated enhancement of spontaneous release, but not depression of evoked release. Occlusion studies with calcium channel blockers suggested that the group III mGluRs might depress evoked release through inhibition of both N and P/Q, but not R-type calcium channels. We suggest that the concurrent depression of action potential-evoked, and enhancement of action potential-independent glutamate release operate through discrete second messenger/effector systems at excitatory entorhinal terminals in rat brain. Transmitter release at central synapses has two components, that driven by action potentials (APs) invading the presynaptic terminal, and an AP-independent component which reflects quantal release. AP-dependent release is often multi-quantal, and depends on calcium entry through voltage-gated calcium channels , we have previously shown that group III mGluRs enhanced the spontaneous release of glutamate . This un2/5% CO2, at room temperature. Following a recovery period of at least 1 h, individual slices were transferred to a recording chamber mounted on the stage of an Olympus  or Zeiss Axioskop FS  upright microscope. The chamber was continuously perfused with oxygenated ACSF at 30–32 °C, at a flow rate of approximately 2 ml/min. The ACSF contained the following (in mM): NaCl (126), KCl (3.25), NaH2PO4 (1.25), NaHCO3 (24), MgSO4 (2), CaCl2 (2.5), and d-glucose (10). The solution was continuously bubbled with 95% O2/5% CO2 to maintain a pH of 7.4. Neurones were visualized using differential interference contrast optics and an infrared video camera.Combined EC–hippocampal slices were prepared from young male Wistar rats (50–110 g) as previously described . All exp2 (0.34), ATP (4), GTP (0.4). The solution was adjusted to 290 mOsmol with sucrose and to pH 7.4 with CsOH. Whole-cell voltage clamp recordings were made from neurones in layer V of the medial division of the EC (mEC), using an Axopatch 200B amplifier . The holding potential in all cases was −60 mV. Under these experimental conditions, layer V neurones exhibit sEPSCs mediated by glutamate acting primarily at AMPA receptors (see N-methyl-d-aspartate (NMDA)-receptor-mediated sEPSCs also occur at a much lower frequency  and had open tip resistances of 4–5 MΩ. They were filled with a solution containing the following (in mM): Cs-methanesulphonate (130), Hepes (10), QX-314 (5), EGTA (0.5), NaCl (1), CaCltors see . Pure N-requency , but in eEPSCs were elicited by electrical stimulation  via a bipolar tungsten electrode placed on the surface of the slice in layer V of the lateral EC. Evoked responses in the mEC can vary greatly in peak amplitude, and from trial to trial, so we adjusted stimulation intensity to always give a sub-maximal (approx. 2/3 maximum amplitude) response, and used of trains of eEPSCs to mitigate single trial variability. For paired-pulse and high frequency trains, an inter-pulse interval of 50 ms was used. For all protocols, stimulation was applied once every 30 s for the duration of the experiment. sEPSCs (consisting of both AP-dependent and independent events) were recorded continuously between eEPSCs. Access resistance was monitored at regular intervals and cells were rejected if this parameter changed by more than ±15%.t-test was used for comparison of eEPSC amplitudes. sEPSCs were detected automatically using a threshold-crossing algorithm, and their frequency and amplitude analyzed. Two hundred sEPSCs were sampled during a continuous recording period for each neurone under each condition. The non-parametric Kolmogorov-Smirnoff (KS) test was used to assess the significance of shifts in cumulative probability distributions of inter-event interval (IEI) (Data were recorded directly to computer hard disk using AxoScope software (Molecular Devices). MiniAnalysis  was used for analysis of both eEPSCs and sEPSCs off-line. For each cell, under each condition, 10 eEPSCs were averaged and peak eEPSC amplitude determined. A paired Student’s al (IEI) . DiffereAll salts used in preparation of ACSF were Analar grade and purchased from Merck/BDH . -1-Aminocyclopentane-1,2,4-tricarboxylic acid (ACPT-1), forskolin and (RS)-cyclopropyl-4-phosphonophenylglycine (CPPG) were obtained from Tocris Cookson . 9-Tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536) was purchased from RBI , tetrodotoxin (TTX) and SNX-482 (SNx) from Alamone Laboratories , and ω-conotoxin GVIA (CTx) and agatoxin IVA (AgTx) from Sigma .P<0.05, KS; n=7). Again, ACPT-1 caused a shift in the distribution of IEI . The mean IEI of mEPSCs was decreased from 666±17 ms to 301±3 ms in the presence of ACPT-1, reflecting a doubling of the frequency. Pooled data from the same seven neurones . The decreased IEI without change in the amplitude of mEPSCs indicated that ACPT-1 increased AP-independent release of glutamate through a presynaptic action, consistent with our previous studies , supporting the conclusion that the effects of the mGluR agonist on IEI were due to presynaptic modulation of glutamate release.We have previously demonstrated that application of the group III mGluR agonist, L-AP4, facilitated spontaneous and AP-independent ‘miniature’ glutamate release (recorded in the presence of TTX) onto layer V neurones . Here, wn of IEI C toward neurones D showed  studies . KineticP<0.05, paired t-test). Having established that the effects of ACPT-1 were mediated by a presynaptic receptor, we next examined the effect of mGluR activation on the evoked component of glutamate release under conditions in which network activity was intact, and both sEPSCs and eEPSCs could be monitored. In recordings from 10 layer V neurones, the mean frequency of sEPSCs was increased during bath application of ACPT-1 (20 μM ACPT-1), as described above and previously reported . ConcurrP>0.05, KS). Similar results were obtained with CPPG at 10 μM (not shown). In contrast, we did observe a robust change in eEPSC amplitude in response to CPPG application. The mean peak amplitude of eEPSCs increased from 48.2±4.5 pA to 57.4±3.1 pA . n=8), the overall profile was initial facilitation, which then plateaued or slightly depressed. CPPG did not alter the profile, but consistently and significantly increased the size of events early in the train. Interestingly, the amplitude of the first event in a train was always facilitated by CPPG, suggesting a constitutive effect via group III mGluRs on AP-dependent release of glutamate at these synapses. In the following experiments we attempted to determine the second messenger events underlying the differential control of glutamate release responsible for sEPSCs and eEPSCs.Given that mGluR activation enhanced spontaneous glutamate release and depressed evoked release, it could be expected that mGluR antagonists might show opposite effects. We applied the group III mGluR antagonist, CPPG (2 μM), while recording both sEPSCs and eEPSCs. B receptors (GABABR) inhibits both AP-dependent and independent GABA release in layer V of the EC, although the intracellular mechanisms have yet to be elucidated , substantially depressed the amplitude of all events within the train. The graphs in P<0.05, t-test). It is clear that the most pronounced effect occurred in the first half of the train reducing the largest events to the extent that an initial facilitation–depression profile of eEPSCs was converted to a gradual facilitation alone , which equates to a percentage reduction similar to that determined previously with ACPT-1 alone. on alone B. It is BRs are strongly activated. The lack of occlusion suggests that group III mGluRs and GABABRs show little convergence at the level of G-proteins or second messengers, and also that GABABRs depress both eEPSC amplitude and sEPSC frequency. We next investigated the second messenger systems underlying the differential modulation of eEPSC amplitude and sEPSC frequency induced by the group III mGluRs.In addition, ACPT-1 appeared to remain effective at increasing the frequency of sEPSCs in the presence of baclofen. P<0.01, KS). However, in the same neurones, the mean amplitude of concurrently recorded eEPSCs was increased from 49.2±4.4 pA to 98.6±8.3 pA in the presence of forskolin (P<0.01). We previously demonstrated that group III mGluR activation enhanced sEPSC frequency by a direct modulation of glutamate release via PKA . PKA hasP<0.01, n=7). However, SQ22536 did prevent the increase in sEPSC frequency elicited by ACPT-1. In order to confirm that the depression of eEPSC amplitude by ACPT-1 was not due to activation of the AC/PKA pathway, we preincubated slices in ACSF containing the specific AC inhibitor, SQ22536 50 μM; , and con2+ in the presynaptic terminals  underlying enhancement of sEPSCs was different to that underlying depression of AP-dependent eEPSCs. Since it is well established that AP-dependent release involves calcium entry through voltage-gated channels, we tested the effect of ACPT-1 on paired-pulse facilitation, a form of short-term plasticity that involves an increase in an evoked response as a result of a preceding conditioning stimulus. It is specific to the set of afferent inputs excited by the first stimulus and is widely believed to depend on residual Caals e.g. . Paired-als e.g.  using anals e.g. A. In sev2+-influx may underlie the ACPT-1-mediated suppression of AP-dependent glutamate release. CTx (1 μM) was used to block N-type channels, and AgTx (200 nM) and SNx (1 μM) were used to block P/Q and R-type channels, respectively. We conducted our toxin experiments in the presence of ascorbic acid (450 μM), since this has been reported to greatly reduce the concentrations of channel toxins required for effective blockade  in the presence of CTx, and to 0.76±0.04 by AgTx (P<0.01). Addition of ACPT-1 saw further reductions to 0.49±0.02 and 0.52±0.03, respectively (P<0.01 in both cases). What is clear is that ACPT plus either toxin results in a total reduction similar to that seen with ACPT alone  reduced the amplitude of the eEPSCs, but this effect was weak, was largely confined to the early, facilitated responses in the train (P<0.01), representing a 50% decrease in amplitude compared with the toxin alone.In a third set of experiments we investigated the possible contribution of R-type VGCCs to the effect of group III mGluRs. In the presence of the tarantula toxin, SNx (1 μM), which is thought to be specific for R-type channels , the abihe train Cb. Indeehe train Cc. Howevhe train Cb. This 2+ (50 μM) to block R-type channels instead of SNx, and saw very similar results. In this case, the mean fractional amplitude in the presence of the divalent cation was 0.84±0.03, but in the presence of ACPT-1 the additional reduction to 0.32±0.01 appeared to be more pronounced (P<0.01), reflecting a reduction by the agonist of approximately 62%. This could be construed as a potentiation of the effects of ACPT-1 by R-channel blockade.In a further set of experiments D we usedonounced Db, c P<02+-influx underlying glutamate release at these terminals, and to approximately the same extent (25–30%). The contribution of R-type channels to evoked release is difficult to assess definitively using current pharmacological tools, since SNx failed to significantly affect eEPSCs overall, while Ni2+ induce a weak depression. However, in some cells , the combination of toxins completely abolished the eEPSCs, showing that simultaneous inhibition of P/Q or N-type VGCCs reduced the intraterminal Ca2+-transient to such a degree that release could not be initiated in most cases. In the remaining neurone  mGluRs can depress glutamate and GABA release independently of Ca2+-channel inhibition . However, recent studies have suggested that the proteins involved in vesicle docking and fusion may be different for the two forms of release  during repetitive stimulation indicates that they are constitutively activated to dampen glutamate release at low frequency, and to limit frequency facilitation. The functional role of the enhancement of activity-independent release by group III receptors is less obvious. We have previously shown that this glutamate-induced glutamate release does seem to be functionally active in the EC. Thus, neurokinin receptor agonists dramatically increase sEPSC frequency in layer V neurones, and this effect is partially ameliorated by a group III antagonist , indicat